The present invention relates to an electrophoresis method, an electrophoresis device, and a marker sample used for the same. More particularly, the present invention relates to an electrophoresis method and an electrophoresis device for separating and detecting a sample labeled with two or more luminescent reagents having different luminescent wavelength ranges, and to a marker sample used for such method and device.
Various devices for detecting a sample by utilizing luminous phenomenon (e.g., fluorescence, chemiluminescence and fluorescent chemiluminescence) are known, such as a fluorescence measuring device, a chemiluminescence measuring device, an electrophoresis device and a biochip reading device. When such devices are used to determine a sample labeled with two or more luminescent reagents with different luminescent wavelength ranges, optical interference filters having high wavelength range selectivities are used for color separation. However, if the wavelength ranges of the luminescent reagents, even partially, overlap each other, a light component of an untargeted luminescent reagent passes through the optical filter, causing leak that needs to be excluded for the subsequent evaluation.
FIG. 1 is a schematic view showing an exemplary device for detecting a sample 2 labeled with two or more luminescent reagents with different luminescent wavelength ranges by using optical interference filters with high wavelength range selectivity. The sample 2 labeled with luminescent reagents is carried on a substrate 1. The light emanating from the luminescent reagents labeling the sample 2 is collected by a condenser lens 3, then transmitted through an optical filter 4, and focused by a convergence lens 5 to a photomultiplier 6 to be detected. The detected signal from the photomultiplier 6 is amplified by an amplifier 7, converted into a digital signal by an A/D converter 8, and processed with a data processor 9.
The optical filter 4 of the device shown in FIG. 1 transmits only a light component within a wavelength range of a luminescent reagent labeling a targeted molecule, and eliminates light components in other wavelength ranges derived from the other luminescent reagents. For sample 2 containing molecules labeled with two or more luminescent reagents with different wavelengths, each molecule may be separately detected by changing the optical filter 4 that allows transmission of light emanated from the luminescent reagent to be detected.
However, the wavelength ranges of luminescent reagents that are actually used often overlap each other. Particularly, when a plurality of (three or more) luminescent reagents are used, it is difficult to select a combination of luminescent reagents such that it does not cause overlapping of their wavelength ranges.
FIG. 2 is a diagram showing wavelength patterns of luminescent light obtained when three types of fluorescent dyes (i.e., fluorescein 14, TMR (carboxy-tetramethyl-rhodamine) 15 and CXR (carboxy-X-rhodamine) 16) are excited with excitation light 13 of 532 nm. The horizontal axis 11 represents wavelength which becomes longer towards right while the vertical axis 12 represents luminous intensity. Generally, when a plurality of luminescent reagents are used as labels, their wavelength ranges overlap as shown in FIG. 2. Thus, even if an optical filter is used for the purpose of obtaining only the light component from the luminescent reagent of interest, light components from other luminescent reagents may leak and pass through the optical filter.
FIG. 3 is the same diagram as that shown in FIG. 2 showing wavelength patterns of luminescent light obtained when the above-mentioned three types of fluorescent dyes are excited with excitation light 13 (532 nm). For example, when an optical filter that transmits light in a wavelength range 17 shown in FIG. 3 is used in a detection system to obtain a light component 16 emanated from the fluorescent dye CXR (hereinafter, referred to as xe2x80x9cCXR light componentxe2x80x9d), a light component 15 emanated from the fluorescent dye TMR (hereinafter, referred to as xe2x80x9cTMR light componentxe2x80x9d) partially overlaps the wavelength range 17 as leakage 18 of the light component 15 through the optical filter for detecting the light component 16. The leakage of a light component of a luminescent reagent other than the luminescent reagent of interest causes detection of a band that is absent in one-dimensional electrophoresis, or detection of a band intensity greater than the band intensity originally obtained in one-dimensional electrophoresis.
FIG. 4 is a diagram illustrating that a measured waveform is deformed due to a leak of an irrelevant light component. Due to the leak of the TMR light component, as shown in FIG. 4, the waveform (electrophoresis pattern) 43 of the CXR-labeled molecule obtained by using the optical filter for extracting CXR light component is deformed from a waveform 41 obtained by one-dimensional electrophoresis of the CXR-labeled molecule. Suppose that the waveform pattern 41 of the CXR-labeled molecule obtained by one-dimensional electrophoresis has two peaks 44 and 45, and the waveform pattern 42 of the TMR-labeled molecule has two peaks 46 and 47. If the TMR light component leaks through the optical filter for extracting CXR light component, the detected electrophoresis pattern is influenced as shown in FIG. 4. Where the molecular weight of the CXR-labeled molecule approximates the molecular weight of the TMR-labeled molecule, the peaks obtained by electrophoresis of both molecules by using the optical filter for detecting CXR overlap each other (peaks 45 and 46) and the electrophoresis pattern 43 gives a peak 48 which is greater than its actual peak 45. Where the TMR-labeled molecule is present and the CXR-labeled molecule is absent, a peak 49 appears on the electrophoresis pattern 43 as influenced by the leakage of light component at peak 47 where there should be no peak.
Such misdetection caused by the leakage of light component emanated from a luminescent reagent other than the luminescent reagent of interest is conventionally corrected by software means. Such software calculates the leakage value, and subtracts that value from the actually measured value. First, positions where or time when a molecule labeled with a luminescent reagent A is solely present are empirically predetermined. Then, values at these positions or time as measured with an optical filter a that transmits light emanated from luminescent reagent A and values at the same positions or time as measured with an optical filter b that is not intended to transmit light emanated from the luminescent reagent A are determined. Based on these values, a leakage rate Rab of the light component emanated from the luminescent reagent A leaking through the optical filter b is calculated. The leakage values at the predetermined points are determined based on the value measured with the optical filter a and the leakage rate Rab . Each leakage value is then subtracted from the value measured at the same point with the optical filter b, thereby eliminating the influence of the leak of the light component from the luminescent reagent A through the optical filter b.
Hereinafter, the process will be described in more detail with reference to FIGS. 5 and 6A-6C. Molecules 53 and 54 are labeled with luminescent reagents A and B, respectively, the luminescent reagents emitting light having different but partially overlapped wavelength ranges. Then, the molecules 53 and 54 are simultaneously but separately subjected to one-dimensional electrophoreses. FIG. 5 is a diagram showing waveforms 51 and 52 measured with the optical filters a and b for detecting light components from luminescent reagents A and B, respectively. The optical filters a and b have selectivity towards the wavelength ranges of the luminescent reagents A and B, respectively. Provided that the molecule 53 labeled with the luminescent reagent A is not detected at the same time as the molecule 54 labeled with the luminescent reagent B, the molecule 53 is detected with the optical filters a and b as a true peak 55 and as a leakage peak 56, respectively. Provided that the molecule 54 labeled with the luminescent reagent B is not detected at the same time as the molecule 53 labeled with the luminescent reagent A, the molecule 54 is detected with the optical filters a and b as a leakage peak 57 and a true peak 58, respectively.
With regard to the molecule 53 labeled with the luminescent reagent A, a leakage rate Rab of the light component from the luminescent reagent A leaking through the optical filter b is determined as a ratio of a peak component 62 (a volume of measured value exceeding a background value 59 measured with the optical filter a) of peak 56 to a peak component 61 (a volume of measured value exceeding a background value 60 measured with the optical filter b) of peak 55. With regard to the molecule 54 labeled with the luminescent reagent B, the rate of the light component from the luminescent reagent B leaking through the optical filter a is determined as a ratio of a peak component 63 (a volume of measured value exceeding a background value 60 measured with the optical filter a) of peak 58 to a peak component 64 (a volume of measured value exceeding a background value 59 measured with the optical filter a) of a peak 57.
Conventionally, in a software developed for the purpose of calculating the leakage rate by means of user interface, the measured peaks are confirmed on a computer display, and a peak area is selected by the user with a pointing device.
FIGS. 6A-6C are diagrams for illustrating how the background value in the selected peak area is determined by the software. The true peak 61 on the waveform 51 (FIG. 5) as measured with the filter a corresponds to peak 71 in FIG. 6A.
As shown in FIG. 6A, for the peak 71 that is represented as a waveform or as a two-dimensional image on a computer display a peak area 74 is determined by selecting the beginning point 72 and the ending point 73 with the pointing device. The peak area 74 is expanded as shown in FIG. 6B for empirically determined widths 75 and 76, thereby determining an expanded peak area 77 for calculating the background value 59 for the optical filter a.
As shown in FIG. 6C, a set of elements of the values in the expanded peak area 77 measured with the optical filter a are converted into a histogram so that the set of elements are distributed based on their values. The resulting histogram will give an elevation 79 consisting of background values and an elevation 78 consisting of peak values. Since the background elevation 79 made up of lower values forms a relatively clear peak, a peak 80 of the elevation consisting of the lower measured values in the histogram is referred to as a background value Ba of the values measured with the optical filter a. Similarly, a set of elements of the values in the expanded peak area 77 measured with the optical filter b are converted into a histogram, and a peak of the elevation consisting of lower measured values in the histogram is referred to as a background value Bb of the values measured with the optical filter b.
Then, the true peak component 61 (FIG. 5) is calculated by subtracting the background value Ba in the selected peak area 74 obtained above (FIG. 6A) from the value measured with the optical filter a in the same area. The leakage peak component 62 (FIG. 5) is calculated by subtracting the background value Bb in the peak area 74 obtained above from the value measured with the optical filter b in the same area. The thus-obtained leakage peak component 62 is divided by the true peak component 61, thereby obtaining leakage rate Rab of light component generated by luminescent reagent A passing through the optical filter b. The leakage rate Rba of light component generated by luminescent reagent B passing through the optical filter a is also obtained in a similar manner.
The thus-obtained background values Ba and Bb and the leakage rates Rab and Rba are applied to the following Equations (1) and (2) below based on value 51 (Pa) measured with the optical filter a and value 52 (Pb) measured with the optical filter b, thereby obtaining values Ta and Tb that are excluded of light component leakage caused by luminescent reagents B and A.
Pa=(Taxe2x88x92Ba)+(Tbxe2x88x92Bb)xc3x97Rba+Baxe2x80x83xe2x80x83(1)
Pb=(Taxe2x88x92Ba)xc3x97Rab+(Tbxe2x88x92Bb)+Bbxe2x80x83xe2x80x83(2)
Background values and leakage rates may also be determined for the case where three or more luminescent reagents and optical filters corresponding thereto are used for the detection. First, two luminescent reagents having the greatest wavelength range overlap area are chosen. Then, using Equations (1) and (2) above, the light component leakage caused by these luminescent reagents is excluded from the measured value. Among other luminescent reagents, one luminescent reagent is chosen which has the greatest wavelength range overlap area with the wavelength range of one of the first two luminescent reagents. Again, using Equations (1) and (2) above, the light component leakage caused by this luminescent reagent is excluded from the measured value. The once-excluded light component leakage Ta or Tb caused by the once-processed luminescent reagent is replaced with Pa or Pb upon the second calculation.
According to such conventional method, the selection of the locations of the true peak to be detected and the selection of the peak areas thereof require clear understanding of the method and skill to use the software. Moreover, in order to obtain uniform results, the user has to be skilled in such experiment so that appropriate background values and leakage rates are always obtained. The background values and leakage rates are highly dependent on the selection of the locations of the true peak to be detected and the selection of peak areas thereof. This has been a problem, for example, in forensic identification where multi-luminescent reagent color separation is utilized as an objective measurement. The conventional method that requires manipulation of the user upon selections of the peaks and the peak areas prevented complete automation of the system of multi-luminescent reagent color separation including the use of the measured results.
The present invention aims at solving such problem and provides a method for automatically obtaining precise background value and leakage rate in color separation and detection thereof using multiple luminescent reagents.
In order to accomplish the above-mentioned aim, the electrophoreosis method of the invention employs a known sample (marker sample) labeled with the same luminescent reagents as the luminescent reagents used for labeling a sample to be subjected to separation measurement. The marker sample and the test sample are simultaneously separated and measured by electrophoresis with a separation device under the same conditions. Since the marker sample can easily be identified from the measured result, a precise background value of the measured value can be calculated. The use of the marker sample allows calculation of precise leakage rate of a light component from a luminescent reagent other than the luminescent reagent of interest. According to the present invention, the background value and the leakage rate of the marker sample upon electrophoresis band measurement can automatically be calculated, and also the leakage light component is automatically subtracted from the measured value.
The electrophoresis method of the invention includes simultaneously electrophoresing a test sample containing a plurality of molecules labeled with a plurality of luminescent reagents and a marker sample containing a plurality of molecules with known molecular weights labeled with the same plurality of luminescent reagents.
Upon such method, a light component emanated from a first luminescent reagent labeling a marker molecule is measured, by using a first optical filter for separating and detecting the light component from the first luminescent reagent, and by using a second optical filter for separating and detecting a light component from a second luminescent reagent, the results being compared with each other, thereby obtaining a leakage rate of the light component from the first luminescent reagent leaking through the second optical filter. This leakage rate is used to correct the measured values of the light components from the respective luminescent reagents.
The plurality of molecules contained in the marker sample are assigned, based on their molecular weights, to a plurality of bands formed by electrophoresis of the marker molecules.
To obtain the leakage rate, peak areas are subtracted from a waveform measured along the electrophoresis distance of the marker sample, and the obtained value is averaged to be used as a background value.
The marker sample of the invention includes various types of molecules having different molecular weights, and various types of luminescent reagents, wherein molecules of the same molecular weights are labeled with the same luminescent reagent.
The marker sample of the invention includes a plurality of marker groups including various types of molecules having different molecular weights, wherein molecules belonging to the same marker group are labeled with the same luminescent reagent, and molecules belonging to different marker groups are labeled with different luminescent reagents. The marker sample of the invention also includes various types of substances such that they are separated at different locations without overlapping each other upon electrophoresis, wherein the various types of substances are grouped into a plurality of groups, and substances belonging to the same group are labeled with the same luminescent reagent, and substances belonging to different groups are labeled with different luminescent reagents.
An electrophoresis device of the invention includes a test sample electrophoresis section where a test sample containing molecules labeled with a plurality of luminescent reagents is electrophoresed and a marker sample electrophoresis section where the above-described marker sample containing molecules labeled with the same plurality of luminescent reagents is electrophoresed. This electrophoresis device may be a slab gel one-dimensional electrophoresis device, a slab gel two-dimensional device, or a a capillary electrophoresis device.
This specification includes all or part of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 10-345476, which is a priority document of the present application.